View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Directory of Open Access Journals

Malaysian Journal of Microbiology, Vol 6(2) 2010, pp. 133-139

Studies on nodulation, biochemical analysis and protein profiles of isolated from species

Kumari B. S., Ram M. R.* and Mallaiah K. V.

Department of Microbiology, Acharya Nagarjuna University Nagarjuna Nagar-522 510, Guntur Dist, Andhra Pradesh, India. E-mail: [email protected] Received 3 July 2009; received in revised form 6 November 2009; accepted 20 November 2009 ______

ABSTRACT

Nodulation characteristics in five species of Indigofera viz., I .trita , I. linnaei , I. astragalina , I. parviflora and I. viscosa was studied at regular intervals on the plants raised in garden soil. Among the species studied, highest average number of nodules per plant of 23 with maximum sized nodules of 8.0 mm diameter was observed in I. astragalina. Biochemical analysis of root nodules of I. astragalina revealed that the leghaemoglobin content of nodules and nitrogen content of root, shoot, leaves and nodules were gradually increased up to 60 DAS, and then decreased with increase in age. Rhizobium isolates of five species of Indigofera were isolated and screened for enzymatic activities and total cellular protein profiles. All the five isolates showed nitrate reductase, citrase, tryptophanase and catalase activity while much variation was observed for enzymes like gelatinase, urease, caseinase, lipase, amylase, lysine decarboxylase and protease activities. Among the isolates studied, only the isolate from I. viscosa has the ability to solubilize the insoluble tricalcium phosphate. All the Rhizobium isolates exhibit similarity in protein content, except the isolate from I. viscosa which showed one additional protein band.

Keywords: Rhizobium, Indigofera species, biochemical analysis, SDS-PAGE ______

INTRODUCTION In general, legumes’ growing wild in any region show adoptability to the environment and fix the atmospheric Indigofera is one of the major nodulated genera in the nitrogen effectively than the cultivated legumes in that family leguminosae. Indigofera was listed as one among region. Recently, the use of effective from wild the nine important legume genera used as green manure, non crop legumes as a bioinoculant to crop plants was with high nitrogen content of 5 and 40 kg N/ha in the proven to increase nodulation and nitrogen fixation leaves (Thonnissen et al., 2000) in wetland rice cultivation (Zahran, 2001). Hence in the present study, five (Allen and Allen, 1981). The species mainly used as green Indigofera species growing wild in our area were screened manure include I. astragalina , I. hirsuta , I. suffruticosa , I. for effective rhizobial strain by comparing the data on tinctoria , I. trita , I. tesymani and I. viscosa . These legumes nodulation; biochemical characteristics and total cellular improve soil texture and tilth, and are important for soil protein profiles of the Rhizobium isolates. conservation and reclamation. Among plant-microbe interactions, legume– MATERIALS AND METHODS Rhizobium interactions are unique because they supply 80-90% of total nitrogen requirement of legumes. It For nodulation studies, seeds of five Indigofera species involves a complex interaction among host, microbial viz., I. trita , I. linnaei , I. astragalina , I. parviflora and I. symbiont and environment. Among nitrogen fixing viscosa were raised in earthen pots using lateritic soil from systems, legume-rhizobium symbiosis is one of the most the experimental plots of the university. Three pots were promising and the bacterial species of Rhizobium complex maintained for each species. The pots were watered are very important (Sprent, 2001). regularly and each pot was maintained with at least 10 Out of the identified 700 species of Indigofera, only plants. Date of nodule initiation for all the five species was 225 species were reported to be nodulated so far. Though recorded by observing the root system of one plant for nodulation was reported in many species of Indigofera , each species, daily after 5 days from the day of sowing. the microsymbiont was isolated and characterized in only Five plants belonging to each species of Indigofera were few species. One species of Rhizobium indigoferae (Wei gently uprooted for studies on nodulation characters like et al ., 2002) from three Indigofera sp. and a species of size, shape, color, number, distribution, fresh weight, dry Sinorhizobium terangae and Bradyrhizobium japonicum weight and moisture content of each nodule. This from I. brevidens (Yates et al. , 2004) were reported so far. nodulation data was recorded at 10 days intervals from 30 to 90 days after sowing. 133

*Corresponding author Mal. J. Microbiol. Vol 6(2) 2010, pp. 133-139

Among the five species, the species with highest number Lysine decarboxylase (4.1.1.18) activity was studied of nodules/plant and biomass was selected for by inoculating the test isolate test tubes containing 10 mL biochemical studies. Biochemical constituents like of Bromocresol purple Falkow medium followed by leghaemoglobin, total nitrogen content of the nodules, incubation at room temperature for 12 h. If the medium leaves, root and shoot were estimated by collecting the turns yellow in response to glucose fermentation in the plant samples at 10 days intervals from 30 to 90 days first 12 h of incubation and remains bright purple, it was after sowing. Leghaemoglobin content was estimated considered as positive test. according to the method described by Tu et al. (1970). Amylase (3.2.1.1) activity was studied by growing the Nitrogen content of the samples as percent dry weight test isolates on Starch Agar Medium (SAM) plates. After was estimated using micro-Kjeldahl ‘N’ method given by incubation at 30 °C for 24 to 48 h., the plates were flooded ANON (1978). The data was statistically analyzed using with iodine solution and observed for starch hydrolysis ANOVA (one way classification technique). around the colony growth e.g. the clear zone. The Rhizobia were isolated from the freshly collected Urease (3.5.1.5) activity was tested by growing the healthy root nodules of all the five Indigofera species on to test organisms on Urea medium plates (20% urea YEMA medium. The identity of the isolates as Rhizobium aqueous solution aseptically added to the basal medium was established by characterization tests including Gram after filtration through Whatman filter paper) containing staining, growth on YEMA medium with Congo red, acid phenol red as pH indicator. Change in color of medium production, ketolactose test, growth in Hofer’s alkaline from yellow to pink was taken as positive test for urease broth, growth on Glucose–peptone agar and nodulation production. test (Holt et al. , 1994).. After confirmation as Rhizobium For the Rhizobium isolates, catalase (1.11.1.6) strains, pure cultures maintained on basal media were activity was tested by growing them on YEMA medium. used in the study. All the five test isolates were screened After incubation for 3 days, 3% H 2O2 was added over the for their ability to produce 13 different enzymes involved in culture. Appearance of effervescence within 20 s indicates biochemical reactions by following standard methods positive catalase activity. (Dubey and Maheshwari, 2002). In all the tests, the The test isolates were streaked across the Sodium isolates were inoculated in to media (or) broth using a pre- Citrate amended YEMA medium plates (Mannitol in YEMA sterilized inoculation loop. was replaced by 1% sodium citrate) added with bromothymol blue indicator. After incubation at room Tests for enzymatic activity temperature for 24–48 h the change in the color of the medium from green to blue indicates citrase (4.1.3.6) Tryptophanase (4.1.99.1) activity was tested by activity. inoculating the test isolates into 1% tryptone broth and To test the gelatinase (3.4.24) activity test tubes incubated at 28 °C for 24-48 h. After incubation, Kovac’s containing 20 mL of gelatin medium (12%) were reagent was added, development of pink color is inoculated with 0.3 mL of test isolate suspension and considered as positive result for tryptophanase incubated for one week. After incubation, the tubes were activity/indole production and absence of color indicates kept in a refrigerator for 30 min and observed for gelatin negative result. liquefaction. All the tests Rhizobia were inoculated on to Protease (3.4.24.25) activity was studied by Skim milk agar (HIMEDIA) plates and incubated at room inoculating the test isolates into YEM broth and incubating temperature for 24–48 h for caseinase activity. Formation at room temperature. During the 6 days of incubation, the of clear zone around the bacterial growth after incubation protease activity was determined at every 24 h. To the 0.2 was considered as positive test. To test the lipase mL of culture suspension, 1 mL of 1% albumin was added (3.1.1.3) activity, plates with YEMA medium supplemented and incubated for 1 h at room temperature. After with 1% Tween-80 were inoculated with tested strains of incubation, 1 mL of 12% Tri-chloro acetic acid (TCA) Rhizobia . The inoculated plates were incubated at 28–30 solution was added and the mixture was cooled rapidly in °C for one week and observed for zone of hydrolysis the ice. The mixture was centrifuged and protein in the around the colony growth. To test the phenylalanine precipitate was estimated by Biuret method. Control was deaminase (4.3.1.24) activity, Rhizobium isolates were maintained by taking 1 mL of albumin added with 1 mL of inoculated on to plates with Phenylalanine agar medium 12% TCA. After mixing the contents thoroughly 0.2 mL of and incubated. After incubation at 30 °C for 48 h, 10% culture suspension was added. If the sample contains FeCl 3 was added and examined for the change in colour. lower protein than the control, it indicates protease Appearance of green color indicates phenylalanine activity. deaminase positive. To study the DNase (3.1.21.1) Nitrate reductase (1.7.99.4) activity was studied by activity, media plates were prepared by using YEMA inoculating 0.5 mL of isolate suspension in to 10 mL of the medium after adding 0.2% DNA at 50 °C. The test YEM broth containing 1% KNO 3 and incubated for 5 days organisms were streaked on the YEMA medium at room temperature. After incubation, 0.1 mL of test containing 0.2% DNA. After incubation for 1–2 days at reagent was added to the test tubes. Development of red room temperature, when the plates were flooded with 1N color within minutes was considered as positive and HCl, DNase activity results in a clear zone surrounded by absence of color indicates negative test. turbidity produced by the precipitate of the unaffected substrate. 134

Mal. J. Microbiol. Vol 6(2) 2010, pp. 133-139

Table 1: Comparative account on root nodulation characteristics of five Indigofera species

Name of the DAS Average Number of Size in Shape Colour *Weight of each *Moisture Plant Nodules/Plant (mm) nodule (mg) content (%) Tap Lateral *Total Fresh Dry root root weight weight I. trita 60 5 4 9 2.0-5.0 Elongated Cream 10.4 3.6 65.3 I. linnaei 70 3 3 6 2.0-7.0 Aggregate Cream 10.2 2.8 72.5 I. astragalina 70 12 11 23 2.0-8.0 Aggregate Cream 16.7 5.1 69.4 I. parviflora 60 4 4 8 1.0-5.0 Globose Pink 3.0 1.2 60.0 I. viscosa 70 4 7 11 2.0-5.0 Elongated Cream 5.8 1.3 77.5 DAS: Days after sowing at which maximum no. of nodules were recorded. Each data is an average of five plants. * significant at 5% between the cultivars (Fc = 12.5 , Ft = 2.603)

Litmus milk reaction Nodulation studies

Test isolates were inoculated into 10 mL of litmus milk The number of nodules increased with age of the plants medium and poured in separate tubes and incubated at and highest number of nodules was observed at 60 DAS 30 °C for 24–72 h. The characteristic reactions including in I. trita and I. parviflora and it was at 70 DAS in I. linnaei , acid production, alkali production, proteolysis and litmus I. astragalina and I. viscose (Table 1). Among the species reduction with serum zone formation were observed after studied the highest average number of nodules per plant incubation. was observed in I. astragalina (23) followed by I. viscosa (10) and a lowest of 6 was observed in I. linnaei . Much Phosphate solubilization test variation in nodule number was reported in Indigofera species. In I. cardifolia , I. hochstetteri and I. linifolia All the five isolates of Indigofera species were also tested highest of only 1–5 nodules per plant reported from for solubilization of tri-calcium phosphate on Pikovskaya’s Pakistan (Athar and Shabbir, 2008). In I. zollingeriana the medium using spot inoculation method (Pikovskaya, highest number of 25 nodules per plant was reported 1948). After 5–7 days of incubation the Pikovskaya’s (Anegbah et al ., 2003). In Indigofera sp (EAO 19), I. medium plates were observed for the formation of clear parniculata and I. pulchra the maximum number reported zone of phosphate solubilization around the colonies. was 29, 24 and 19 nodules per plant, respectively Analysis of total cellular proteins of rhizobia isolated (Ezedinma et al ., 1978). In the present study, the size of from all the five Indigofera species was carried out by the nodules ranged from (2.0–8.0 mm), the maximum SDS-PAGE by method given by Laemmli (1970). After the nodule diameter observed was in I. astragalina followed electrophoresis, similarity index (percentage similarity by I. linnaei (7 mm) and in rest of the species it was between the isolates) was calculated dividing the total observed as 5 mm. The root nodules of Indigofera are number of similar bands with total number of bands, round to oval type at the early stage but became multiplied by 100. elongated with increase in age. The nodules are globose and pink in I. parviflora , while the nodules of I. astragalina RESULTS AND DISCUSSION and I. linnaei are cream and aggregated. The fresh weight of each nodule was more in I. astragalina (16.7 mg) All the Indigofera species studied showed variation in and minimum in I. parviflora (3.0 mg). The dry weight of nodulation characteristics. Among the species studied, each nodule was also more in I. astragalina (5.1 mg), and nodulation was initiated much earlier at 22 DAS in case of minimum in I. viscosa and I. parviflora (1.3 mg and 1.2 mg, I. trita and I. astragalina, where as in I. viscosa , I. respectively). Maximum moisture content of 77.5% in the parviflora and I. linnaei nodulation started at 28, 30 and 35 nodules was observed in I. viscosa while only of 60% was DAS respectively. The nodulation in the present study can recorded in I. parviflora. The statistical analysis by taking be referred as diffused type as they are distributed both cultivars as one treatment and the other parameters like on tap and lateral roots, with more on tap root than on no. of nodules, fresh and dry weight of the nodule as the lateral roots. Bhaduri and Sen (1968) classified the pattern second treatment reveals the F-calculated (Fc) value was of nodulation in Phaseolus and Glycine max in to three greater that F-tabulated (Ft) value. If Fc>Ft then it is distinct types. Localized e.g. on the tap root only; diffused considered as significant. Therefore significance exists e.g. nodules diffused in tap and lateral roots and Mixed between cultivars and other parameters. e.g. showing a tendency towards grouping of a few In all the Indigofera species studied, nodulation was nodules in the tap root, the rest being diffused in the initiated 3 weeks after seed germination and the number laterals. Similar pattern of diffused type of nodulation was increased with age of the plant up to 70 DAS. That the reported in Indigifera zollingeriana by Anegbah et al. number of nodules mainly increased during vegetative (2003) and in Phaseolus sp. by Bhaduri and Sen (1968). phase (30 to 70 d) and decline during flowering and pod

135

Mal. J. Microbiol. Vol 6(2) 2010, pp. 133-139

second treatment reveals the F-calculated (Fc) value was greater that F-tabulated (Ft) value. If Fc>Ft then it is considered as significant. Therefore significance exists between nitrogen contents and different plant parts.

Table 2: Biochemical analysis of different plant parts of Indigofera astragalina at different growth stages

DAS Leghaemo- *Nitrogen content (%) globin content Nodules Leaves Root Shoot (µg/g) 30 312 1.68 0.56 0.56 0.56 40 328 2.52 1.12 0.70 3.08 50 424 3.36 1.12 0.84 1.40 60 512 4.76 1.40 0.98 1.26 70 390 3.92 1.26 0.70 0.84 80 216 1.26 0.84 0.56 0.56 90 171 1.40 0.56 0.56 0.42 Figure 1: Electrophoretic banding pattern of total cellular proteins of five Rhizobium isolates from DAS days after sowing Indigofera species Each data is an average of three replicates. *Nitrogen content was significant at 5% between different plant parts (Fc = 8.02, Ft = 3.00) setting phase, was also reported by Arya and Singh (1996) in horse gram. Identification of Rhizobium strains

Biochemical studies From the characterization tests it is evident that all the isolates form five Indigofera species are gram negative The leghaemoglobin content of I. astragalina nodules rods; acid producers; non 3-ketolactose producers and gradually increased with age of the plant from 30 DAS and showed no growth in Hofer’s medium and Glucose– reached maximum of 512 µg/g at 60 DAS (Table 2). The peptone agar and are finally confirmed as Rhizobia by the leghaemoglobin content then showed a sudden decrease nodulation test. and reached the minimum of 171 µg/g at 90 DAS. A gradual increase in nitrogen content was also observed Enzymatic studies along with increase in leghaemoglobin content of the nodules and reached the maximum at 60 DAS. Similar The ability to produce different enzymes to utilize various type of positive correlation was also reported by Sidhu et organic substrates is an important biochemical al. (1967) in 6 legumes and Subba Rao and Chopra (1967) characteristic feature of Rhizobium strains . Among the in soyabean. Raghava et al. (1993) reported that enzymatic activities studied, all the five isolates showed leghaemoglobin content of the nodules was peaked at 40– tryptophanase, nitrate reductase, citrase and catalase 45 days. However, in the present study the peak was activities and do not exhibit phenyl alanine deaminase and observed at 60 DAS. DNase activities (Table 3). Variation was observed among Among the different plant parts analyzed, the the isolates for the enzymatic activities like protease, maximum nitrogen content recorded was 4.76% in urease, gelatinase, caseinase, lipase, amylase and lysine nodules, followed by leaves with 1.40% and shoot 1.26 % decarboxylase. The variation in enzymatic activities of at 60 DAS. The nitrogen content of nodules was 3 times rhizobial isolates was reported by various workers. higher than that recorded in other plant parts at 30 DAS Graham and Parker (1964) reported that out of 79 strains and showed a rapid increase in nitrogen content with of rhizobia studied, 58 are positive to nitrate reduction increase in age. In all the aerial parts, the nitrogen content while 21 are negative. In the present study 4 out of 5 increased gradually from 30 DAS and reached maximum isolates were positive to nitrate reductase activity. Salve at 60 DAS and showed gradual decrease afterwards. and Gangawanae (1992) reported that out of the 13 Ezedinma et al. (1978) reported that among the 15 isolates studied by them some are positive and some are species of Indigofera studied, Indigofera sp. (EAO 19) negative. In the present study only the isolate from I. trita gave the highest nitrogen concentration in the leaves showed amylase activity. Nitrate reductase activity, (4.27%) followed by I. simplicifolia , I. hirsuta and I. production of ammonia and catalase activities play an parniculata . In the present study, relatively high nitrogen important role in nitrogen fixation metabolism. Enzymes content was observed in nodules and leaves than in shoot, like urease, protease, amylase, gelatinase play an supporting the green manure status of I. astragalina . The important role during nodule formation. Thus, the statistical analysis by taking nitrogen at different DAS as Rhizobium isolates which produce these enzymes are one treatment and N content in other plant parts as the considered as best for nodulation and nitrogen fixation.

136

Mal. J. Microbiol. Vol 6(2) 2010, pp. 133-139

Table 3: Enzymatic/Biochemical activities of Rhizobium isolates of Indigofera species

Enzymatic activities tested Name of the isolates I. trita I. linnaei I. astragalina I. parviflora I. viscosa Tryptophanase + + + + + Nitrate reductase + - + + + Lysine decarboxylase + + - - + Amylase + - - - - Urease + + - - - Catalase + + + + + Citrase + + + + + Gelatinase - + + - + Caseinase - - + - - Lipase - + - - + Protease + + + - + Phenylalanine deaminase - - - - - DNase - - - - - Litmus milk reaction Acid Alkaline Proteolysis Proteolysis Litmus reduction Phosphate solubilization - - - - + (Pikovskaya’s medium)

Much variation was observed among the isolates legumes, exhibited protein profiles with peptide bands with respect to litmus milk reaction. The isolate from I. trita ranging from 5–19 bands per profile was reported by produced acid and the isolate from I. linnaei produced Zahran et al. (2003). In the present study, all the five alkaline. The two isolates from I. astragalina and I. isolates showed two common bands at mol. wt. of 66,000 parviflora showed proteolysis and the isolate from I. Da and 43,000 Da and shared rest of the three bands viscosa showed litmus reduction with serum zone between different isolates. Sridevi and Mallaiah (2008) formation. Growth reactions of Rhizobium in litmus milk, studied the SDS PAGE analysis of whole cell–protein and one of the characterization test to differentiate different reported that Rhizobium isolate from Sesbania sesban isolates, have been studied by various workers (Graham produced a band at 70,000 Da for the outer membrane and Parker, 1964; Muthusamy et al ., 1973; Rangaswami receptor protein involved in siderophore transport. and Oblisami, 1962; Oblisami, 1974) and these studies Similarly in the present study also a common band for all revealed that, the Rhizobium isolates from clover, pea and the isolates was observed just above 66,000 Da, Vicia group produce alkalinity with the production of serum indicating that these isolates are also potential zone. While the rhizobia from cowpea, soyabean and siderophore producers. The isolate from I. viscosa lupin, produce alkalinity without the production of serum showed one additional band with mol. wt. of 29,000 Da, zone. However, Basak and Goyal (1980) reported that 4 which was not shared by any other isolates. The isolates isolates from Acacia and Albizia produce acidity without from I. linnae, I . astragalina and I. viscosa shared a serum zone production. In the present study, the isolate common band with mol. wt. of 14,300 Da. In I. astragalina from I. viscosa produced serum zone with acid reaction. among the five bands, one band with approx. mobility at This indicates that the present Rhizobium isolates from 23,000 Da was shared with that from I trita isolate while Indigofera species appears to be quite different from most another band at approx mobility at 18,000 Da was shared of the Rhizobia , with respect to litmus milk reaction. with that from I. parviflora isolate. The SDS-PAGE Among the five isolates studied the isolate from I. analysis of whole cell proteins not only helps in identifying viscosa only showed clear zone around the colony in of the rhizobial strains (Roberts et al. , 1980; Fabiano and Pikovskaya’s medium, indicating the ability to solubilize tri Arias, 1990) but also useful in the differentiation among calcium phosphate. Thus, phosphate solubilization, an the isolates within the same serogroup (Broughton et al. , important criterion for plant growth promoting 1987). By conducting the SDA-PAGE analysis of four rhizobacteria, was exhibited by only I. viscosa isolate. Azorhizobium strains isolated from stem nodules of Sesbania rostrata Dreyfus et al. (1985) concluded that all Protein profiles the four strains have identical protein gel electropherograms and are closely related. In the present In the present study, electrophoretic banding pattern of study the similarity index studies based on the number of whole cell protein of five Rhizobium isolates was bands, revealed that the isolate from I. astragalina performed to study the similarity between them. Maximum showed 75% similarity with I. trita , I. linnaei , and I. of five bands were observed in the isolate from I. parviflora , while the isolate from I. viscosa was different astragalina and four in the isolate from I. viscosa , while from other isolates by having an additional band. This rest of the isolates produce only 3 bands (Figure 1). SDS- clearly shows that the isolates were related with reference PAGE of whole-cell proteins of rhizobial strains from wild to number of bands but differ with reference to the quality 137

Mal. J. Microbiol. Vol 6(2) 2010, pp. 133-139

and quantity of the proteins. Zahran et al. (1994) reported as green manure in paddy fields. Mircen Journal 1, a change in SDS PAGE protein banding pattern among 111-121. the salt tolarent (that can grow up to 3% NaCl) and Dubey, R. C. and Maheshwari, D. K. (2002). Practical halophylic strains (that grow in the range of 3 – 10% NaCl) Microbiology. S. Chand and Co. Ltd., New Delhi. pp. of Rhizobia . Similarly, the difference in banding pattern 352. observed in the present study by the presence of an Ezedinma, F. O. C., Agnim, N. N. and Onyekwelu, S. S. additional band in the isolate from I. viscosa can be C. (1978). The contribution of symbiotic nitrogen correlated to the fact that this was the only halophylic fixation to the nitrogen economy of natural isolate among the five isolates that can grow at 6% NaCl ecosystem. Occurrence of herbaceous legumes in concentration (data not presented but studied). Thus, the derived Savanna fallow and their nodulation in pot isolate from I. viscosa with its halophylic nature and culture. Plant and Soil 51, 503-515. phosphate solubilizing capacity, can be exploited as an Fabiano, E and Arias, A. (1990). Identification of effective bioinoclulant. inoculants, strains and neutralized population of Rhizobium leguminosarum bv. Trifolii using ACKNOWLEDGEMENTS complementary methodologies. World Journal of Microbiology and Biotechnology 6, 27-133. Authors are thankful to the Department of Microbiology, Graham, P. H. and Parker, C. A. (1964). Diagnostic Acharya Nagarjuna University, for providing facilities for features in the characterization of the root nodule this work. Saritha Kumari was also thankful to Andhra of legumes. Plant and Soil 20, 383-396. Pradesh State Council of Science and Technology, Holt, J. G., Krieg, N. R., Sneath, P. H. A., Staley, J. T. Hyderabad, for the Young Scientist Fellowship. We are and Williams, S. T. (1994). Bergey’s Manual of also thankful to Prof. G.V.S.R. Anjaneyulu of Statistics Determinative Bacteriology. 9th edn. Williams and department for the statistical analysis in the present study. Wilkins, A. Wavely company, Baltimore. pp. 787. Laemmli, U. K. (1970). Cleavage of structural proteins REFERENCES during the assembly of head bacteriophage T 4. Nature 227, 680-685. Allen, O. N. and Allen, E. K. (1981). The Leguminosae. A Muthusamy, S., Vidhyasekaran, P. and Govindsamy, Source Book of Characteristics, Uses and Nodulation. C.V. (1973). Studies on Rhizobium strains of ground The University of Wisconsin Press, Madison, Wisc. nut. Indian Journal of Microbiology 13, 143-148. U.S.A. pp. 341-351. Oblisami, G. (1974). Studies on Rhizobium and Anegbah, P., Tachoundjeu, Z. and Amakiri, M. (2003). nodulation pattern in a forage legume, Clitoria ternata Characteristics of nodules of Agroforestry tree isolated from paddy field. Indian National Science legumes for sustainable production in Niger Delta Academy Proceeding B 40, 618-623. Region, Nigeria. Improvement and Culture of Pikovskaya, R.I. (1948). Mobilization of phosphorous in Nitrogen Fixing Trees 3(1), 2-3. soil in connection with vital activity of some microbial Annonymous (1978). Official and tentative methods of species. Mikrobiologiya 17, 362-370. analysis. Association of Official Agricultural Chemists. Raghava, N., Raghava, R. P. and Kumar, P. (1993). Washington, D.C. Protein and leghaemoglobin content in nodules in Arya, M. P. S. and Singh, R. (1996). Effect of N, P and K, relation to flowering and pod formation in Vigna on the nodulation, growth and yield characters of unguiculata L. Walp. Indian Journal of Plant. horse gram [Macrotyloma uniflorum (Lam) verdc.]. Physiology 36, 67-68. Legume Research 19, 65-69. Rangaswami, G. and Oblisami, G. (1962). Studies on Athar, M. and Shabbir, S. M. (2008). Nodulating some legume root nodule bacteria. Journal of the leguminous weeds of some major crops of Pakistan. Indian Society of Soil Science 10, 175-186. Phytologia 90(2), 246-251. Roberts, G. P., Leps, W. T., Silver, L. E. and Brill, W. J. Basak, M. K. and Goyal, S. K. (1980). Studies on tree (1980). Use of two dimensions polyacralamide gel legumes III. Characterization of the symbionts and electrophoresis to identify and classify Rhizobium direct and reciprocal cross inoculation studies with strains. Applied and Environmental Microbiology 39, tree legumes and cultivated legumes. Plant and Soil 414-422. 56, 39-51 . Salve, P. B. and Gangwanae, L. V. (1992). Rhizobium Bhaduri, P. N. and Sen, R. (1968). Distribution pattern of from wild legumes and nitrogen fixation in ground nut. nodules in Phaseolus species and Glycine max. In: Biofertilizers Technology Transfers. Associated Indian Journal of Genetics and Plant Breeding 28, Publications Co., New Delhi. pp.95-100. 287-296. Sidhu, G. S., Singh, M. and Singh, R. (1967). Symbiotic Broughton, W. J., Samrey, U and Stanely, J. (1987). nitrogen fixation in some legumes in Punjab. 1. role of Ecological genetics of Rhizobium meliloti : Symbiotic leghaemoglobin in nitrogen fixation. Journal of plasmid transfer in the Medicago sativa rhizosphere. Research Punjab Agriculture University 4, 244-248. FEMS Microbiology Letters 40 , 251-255. Sprent, J.I (2001). Nodulation in legumes. Royal Dreyfus, B., Rinaudo, G. and Dommergues, Y. R. Botanical Gardens, Kew, London. (1985). Observations on the use of Sesbania rostrata 138

Mal. J. Microbiol. Vol 6(2) 2010, pp. 133-139

Sridevi, M and Mallaiah, K. V. (2008). Production of Hydroxymate- type Siderophores by Rhizobium strains from Sesbania senban (L.) Merr . International Journal of Soil Science 3(1), 28-34. Subba Rao, N. S. and Chopra, C. L. (1967). Leghaemoglobin and its role in symbiotic nitrogen fixation. Journal of Scientific and Industrial Research 26, 329-332. Thonnissen, C., Midmore, D. J., Ladha, J. K., Olk, D. C. and Schmidhalter, U. (2000). Legume decomposition and nitrogen release when applied as green manures to tropical vegetable production systems. Agronomy Journal 92, 253-260. Tu, J. C., Ford, R. E. and Garu, C. R. (1970). Some factors affecting the nodulation and nodule efficiency in soyabeans infected by soyabean mosaic virus. Phytopathology 60, 1653-1656. Wei, G. H., Chen, W. X., Wang, E. T., Tan, Z. X. and Zhu, M. E. (2002). Rhizobium indigoferae sp. nov. and Sinorhizobium kummerowiae sp. nov., respectively isolated from Indigofera spp. and Kummerowia stipulacea . International Journal of Systematic and Evolutionary Microbiology 52, 2231- 2239. Yates, R. J., Howeieson J. G., Nandasena K. G. and O’Hara G. W. (2004). Root-nodule bacteria from indigenous legumes in the north Western Australia and their interaction with exotic legumes. Soil Biology and Biochemistry 36, 1319-1329. Zahran, H. H. (2001) . Rhizobia from wild legumes: diversity, , ecology, nitrogen fixation and biotechnology. Journal of Biotechnology 91, 143-153. Zahran, H. H., Rasanen, L. A., Karsisto, M. and Lindstrom. (1994). Alteration of lipopolysaccharide and protein profiles in SDS-PAGE of rhizobia by osmatic and heat stress. World Journal of Microbiology Biotechnology 10, 100-105. Zahran, H. H., Abdel-Fattah, M., Ahmad M. S. and Zaky A. Y. (2003). Polyphasic taxonomy of symbiotic rhizobia from wild leguminous plants growing in Egypt. Folia Microbiologica 48(4), 510-520.

139